Vaccinia virus, the prototypic poxvirus, was used in the landmark vaccination campaign that eradicated smallpox. In the post-vaccination era, monkeypox virus is emerging as a serious human pathogen, and concern about the possible bioterrorist use of variola remains. With the development of poxviruses as recombinant vaccines and effective tools for oncolytic therapy, however, these viruses are once again seen in a promising light. In order to develop rational, targeted therapeutics to treat complications that may arise during exposure to poxviruses, a deeper understanding of the intricacies of the poxvirus life cycle is needed. Poxviruses replicate solely within the cytoplasm of the infected cell. This unusual physical autonomy from the nucleus is accompanied by genetic complexity: ~200 viral proteins regulate viral entry, gene expression, genome replication and maturation, and virion assembly and egress. Despite this genetic autonomy, the close relationship between cell biological processes and the progression of the viral life cycle is increasingly clear. It is he interplay between vaccinia virus infection and the bioenergetic status of the cell that is the focu of this R21 application. Our preliminary data indicate that viral infection depends upon the synthesis and mitochondrial import of palmitate to fuel the TCA cycle and ATP production. Moreover, infection leads rapidly to a ~2-fold increase in the cellular oxygen consumption rate (OCR), a direct measure of ATP production. Although many phases of the life cycle show moderate impairment when fatty acid synthesis is reduced, it is virion assembly that appears to be the most severely impacted. There is a clear impairment of the structure and function of the virosomes, which are the depots of high concentrations of soluble proteins destined for inclusion in the virion interior. The viroplasm appears to become dispersed into aggregated fragments which fail to make appropriate associations with the nascent viral membranes. The proposal is organized into two complementary aims: Aim I: How does vaccinia virus infection modulate fatty acid synthesis and mitochondrial function? A: How are the synthesis and utilization of fatty acids regulated during vaccinia infection? B: How are mitochondrial activity and ATP generation enhanced during infection? Aim II: Why is virion assembly reliant on ATP generation? This aim will test two hypotheses regarding the role of ATP: Is ATP required to ensure the phosphorylation of viral proteins needed for viroplasm stability and association with crescent membranes? Is ATP needed to support the association of cellular chaperones (such as HSP90) with viroplasmic proteins to ensure their proper folding? The insights gained should illuminate a new aspect of poxvirus/host interaction and identify new cellular targets for anti-poxviral therapy Moreover, a deeper understanding of how vaccinia manipulates the cellular bioenergetic environment, and how chaperones or protein kinases regulate the solubility of highly concentrated protein complexes, should be of broad relevance to the study of cancer and neurodegenerative diseases.